Acute myeloid leukemia (AML) is a heterogeneous disease with diverse gene mutations and chromosomal abnormalities. Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for approximately 24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia. The encoded proteins, RUNX1 and CBFbeta, form a heterodimer to regulate gene expression, and they are both required for hematopoiesis in vertebrate animals from zebrafish to man. Extensive clinical studies have demonstrated that CBFB-MYH11 and RUNX1-ETO, the two common fusion genes in CBF leukemia, are the best biomarkers for diagnosis, prognosis, and residual disease monitoring of CBF leukemia patients. Even though CBF leukemias have better initial remission rate and better prognosis than most AML cases, current chemotherapy is associated with significant morbidity and mortality, and the long-term survival (>5 year) is only around 50-60%. Over the years we have used mouse models and a variety of research tools to characterize the CBFB-MYH11 fusion gene, determine the effect of the encoded protein, CBFbeta-SMMHC, on normal hematopoiesis, and understand the leukemogenesis process associated with the fusion gene. We have generated both conventional and conditional knock-in mouse models to study CBFB-MYH11. Using these mouse models we have demonstrated that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB function during definitive hematopoiesis, resulting in total loss of definitive hematopoiesis in the heterozygous Cbfb-MYH11 knockin mouse embryos. We also showed that Cbfb-MYH11 is necessary but not sufficient for leukemia, and we were able to identify cooperating genetic events in the mouse models. We have generated knock-in mouse models expressing truncated Cbfb-MYH11 to determine the importance of functional domains of CBFbeta-SMMHC. Overall our lab has been recognized in the field as the major contributor to the understanding of CBFB-MYH11 leukemia. In the last fiscal year we have continued our studies on the mechanisms of leukemogenesis by CBFB-MYH11. In the first specific aim we determined if RUNX1 is important for leukemogenesis by CBFB-MYH11. Previously dominant negative inhibition of normal RUNX1 and CBF functions has been considered as a potential mechanism for CBF-SMMHC. However, recently we showed that Cbfb-MYH11 knockin embryos have primitive hematopoiesis defects that do not seem to result from RUNX1 repression (Hyde et al., Blood, 2010). Moreover, knockin mice expressing a modified CBF-SMMHC protein with decreased RUNX1-binding ability developed leukemia faster than those that express the full-length CBF-SMMHC (Kamikubo et al., Cancer Cell, 2010). These findings suggested that RUNX1-repression may not be important for leukemogenesis, and raised the possibility that CBF-SMMHC may induce leukemia independent of RUNX1. To test this hypothesis, we have used three Runx1 deficient models to determine if RUNX1 is required for leukemogenesis by CBF-SMMHC. In Cbfb+/MYH11 embryos that are also Runx1-/-, or with a semi-dominant-negative Runx1 allele, Runx1+/lz, the primitive hematopoietic defect induced by Cbfb-MYH11 was rescued, even though Runx1 deficient embryos did not have primitive hematopoietic defects. During definitive hematopoiesis in adults, CBF-SMMHC increased proliferation of progenitor cells and induced an abnormal pre-leukemic progenitor population. These defects were also rescued by the semi-dominant-negative allele, Runx1+/lz, or a conditional Runx1 null. Finally, leukemia development was significantly delayed in Cbfb+/MYH11, Runx1+/lz or Cbfb+/MYH11, conditional Runx1 null mice. Overall, our findings suggest that RUNX1 activity is required for Cbfb-MYH11-induced hematopoietic defects and leukemogenesis. In the second specific aim we studied the potential cooperation between CHD7 and CBFB-MYH11 for leukemogenesis. The chromodomain-helicase-DNA binding protein 7 (CHD7) interacts with RUNX1 and suppresses RUNX1 function during hematopoiesis. We hypothesized that CHD7 also plays a role in leukemogenesis by CBFB-MYH11, since CBFB-MYH11 requires RUNX1 for leukemia. To test this hypothesis, we crossed conditional Chd7 knockout mice (Chd7f/f) with Cbfb-MYH11 knockin mice to generate transgenic mice expressing Cbfbeta-SMMHC but deficient for CHD7. We found that the hematopoietic progenitor cell populations were significantly lower in these transgenic mice than control mice, which was likely due to reduced cellular proliferation. Importantly, it took much longer time for these transgenic mice to develop leukemia than the mice only expressing CBFbeta-SMMHC. We also showed that CHD7 is a partner of the RUNX1-CBFbeta-SMMHC transcription complex and that CHD7 could enhance transcription of RUNX1 and CBFbeta-SMMHCs target genes. These data indicate that CHD7 deficiency inhibits Cbfb-MYH11 induced leukemogenesis through inhibiting RUNX1 activity in regulating transcription and cellular proliferation. In the third specific aim we are studying the interaction between RUNX1 and U2AF1 for leukemogenesis. The pathogenesis of hematopoietic malignancies, including AML and myelodysplastic syndromes (MDS), is very complicated with multiple genetic alterations required for full-blown disease. Mutations of splicing factor genes, including U2AF1, are found in overall half of MDS patients and 510% de novo AML patients. U2AF1 mutations, occurring in roughly 10% of patients with MDS and 3% in de novo AML, are heterozygous and localized almost exclusively in two codons, S34 and Q157, suggesting they are gain-of-function mutations. In addition, our recent studies suggest retention of the wild type U2AF1 allele is required for cell viability. Interestingly, RUNX1 is the most commonly co-mutated gene in MDS patients with U2AF1 mutations, and U2AF1 and RUNX1 are also co-mutated in some AML patients. These findings suggest RUNX1 deficiency and U2AF1 mutation cooperate in the pathogenesis of MDS and AML. To test this hypothesis, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f ) with mice carrying a newly developed Cre-based conditional U2af1 S34F mutation (U2af1+/S34F ) to generate Runx1f/f, Mx1Cre, U2af1+/S34F mice. Interestingly, of fourteen Runx1f/f, Mx1Cre, U2af1+/S34F mice, two developed AML, and the rest developed MDS. Moreover, transplanted spleen and bone marrow cells from one of four MDS Runx1f/f, Mx1Cre, U2af1+/S34F mice also developed AML in the recipients. Although Runx1f/f, Mx1Cre mice also had MDS, their MDS cells did not develop into leukemia after transplantation. Our data therefore suggest that Runx1 deficiency and MDS-associated U2af1 mutation can cooperate in the genesis of AML from MDS.